Mediator-modified redox biomolecules for use in electrochemical determination of analyte

ABSTRACT

Compositions and methods for electrochemical detection of an analyte comprising a transition metal compound 
     
       
         
         
             
             
         
       
         
         
           
             wherein M is a metallic element that can form a coordinate bond to nitrogen; R and R′ are coordinated to M at their nitrogen atoms; L is a linking ligand; Z is chlorine or bromine; m can be from 1 to 6 and X is an anion, or combination of anions, that balances the charge m. Also provided are electrochemical tags and methods of detection.

FIELD OF THE INVENTION

This application relates generally to biosensors.

BACKGROUND OF THE INVENTION

Redox biomolecules undergo reversible reduction and oxidation andefficiently transfer electrons to a natural electron acceptor. Theygenerally contain an active site prosthetic group or cofactor thatmediates and participates in an oxidation and reduction pathway. Mostcommonly, the prosthetic group is based on dinucleotides such as flavinadenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD).The redox biomolecules oxidize and accept electrons from a substrate andthen transfer the electrons by means of a reversible oxidation andreduction of the prosthetic group to an electron acceptor.

Biosensors can provide sensitive, rapid and low cost assays fordetection of analytes in a sample. A biosensor essentially comprises aredox biomolecule that recognizes a target analyte and a transducer thatconverts the recognition event into a measurable signal. In one sense,the biosensor operates by “interrupting” the flow of electrons to thenatural electron acceptor. The flow is detected as a current or voltageby an electrical circuit containing an electrode in proximity to thebiomolecule. The transfer of electrons between the active siteprosthetic group of the redox biomolecule and an electrode surface isthus an important factor in the efficient operation of biosensors.

It is generally observed that efficiency of electron transfer from redoxbiomolecules to the electrode surface of a biosensor is less than thehighly efficient reduction of the natural electron acceptor. Severalgroups have investigated the modification of redox biomolecules bycovalent attachment of a redox mediator, but found the electron transferrate constants to be far lower than those between enzymes and theirnatural electron acceptor.

SUMMARY OF THE INVENTION

In accordance with the present invention, Applicants have successfullydevised novel approaches in biosensors for detecting an analyte in asample. The novel approaches are based on synthesis of novel oxidationreduction mediators that can lose or gain electrons under variousexperimental conditions. It is believed that such redox mediators act aselectron relay groups that enable non-diffusion-mediated electrontransfer from other oxidation reduction molecules such as, for example,oxidoreductases.

In various embodiments, the present invention provides transition metalcompounds having a structure that corresponds to the formula I:

wherein M is a metallic element that can form a coordinate bond tonitrogen. R and R′ can be nitrogen-containing organic moietiescoordinated to M at their nitrogen atoms. L can be a linking ligandcomprising an organic amine having from about 3 to about 20 non-hydrogenatoms, an aliphatic amino group and a nitrogenous moiety that provides ametal-to-nitrogen coordinate bond to M. Z can be a halogen atom and mcan be +1, +2, +3, +4, +5, or +6. X is an anion, or combination ofanions, that balances m.

In various other embodiments, the present invention also providesmethods of preparing transition metal compounds having theabove-described structure that corresponds to formula I. The methodprovides for contacting a precursor compound of the formula II:

wherein Z, M, R, R′, X and m are as described above, with a linkingligand to form the transition metal compound of formula I via a ligandexchange reaction.

In further embodiments, the present invention provides anelectrochemical tag comprising a redox biomolecule bonded to atransition metal compound of formula I wherein Z, L, M, R, R′, X and mare as described above. In a preferred embodiment, M is osmium and L is3-aminopropylimidazole. Preferably, the transition metal compound iscovalently attached to the redox biomolecule, which preferably comprisesglucose oxidase.

In various further embodiments, the present invention also providesmethods for preparing the above-described electrochemical tags. Themethod comprises bonding a redox biomolecule to a transition metalcompound of formula I wherein Z, L, M, R, R′, X and m are as describedabove. In a preferred embodiment, Z is chlorine, L is3-aminopropylimidazole and the transition metal compound is covalentlyattached to glucose oxidase by amide bond formation between glucoseoxidase carboxylates and the aliphatic primary amino group present on L.

The present invention further provides methods for electrochemicaldetection of an analyte in a sample utilizing the above-describedelectrochemical tags. In various embodiments, an electrochemicallyactive redox complex comprising the analyte and an electrochemical tagis formed at the electrode surface, wherein the electrochemical tagcomprises a redox biomolecule bonded to a transition metal compound offormula I wherein Z, L, M, R, R′, X and m are as described above.Without limiting the utility, function, or composition of the presentinvention, the shuttling of electrons between the electrochemical tag,which undergoes cycles of oxidation and reduction, to the electrode canbe detected and is indicative of the presence or absence of the analytein the sample. The analyte to be detected can be, for example, a nucleicacid or a protein. In a preferred embodiment, the analyte is a sequencecorresponding to the p53 gene, which is a gene associated with a varietyof cancers. In other embodiments, the analyte to be detected can also bethe oxidation substrate for the redox biomolecule.

In still further embodiments, the present invention is also directed tokits comprising the above-described electrochemical tags, as well as, tobiosensors that utilize such electrochemical tags for electrochemicaldetection of analytes.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 is an illustration of data describing cyclic voltammetry ofOs(bpy)₂(3-aminopropylimidazole)Cl modified glucose oxidase (GOx)adsorbed on screen-printed carbon electrode in PBS buffer. (a) in thepresence of EDC/NHS coupling agent, (b) in the absence of EDC/NHScoupling agent. Scan rate: 100 mV/s.

FIG. 2 is an illustration of UV-Vis spectra of (a) GOx (b)Os(bpy)₂(3-aminopropylimidazole)Cl and (c)Os(bpy)₂(3-aminopropylimidazole)Cl modified GOx.

FIG. 3 is an illustration of amperometric response ofOs(bpy)₂(3-aminopropylimidazole)Cl modified GOx adsorbed on ascreen-printed carbon electrode. (a) in the presence of EDC/NHS couplingagent, (b) in the absence of EDC/NHS coupling agent. Poised potential:0.30 V, glucose concentration: 40 mM.

FIG. 4 is an illustration of amperometric response of DNA assay (p53gene in mRNA, 20 pg/μl) using Os(bpy)₂(3-aminopropylimidazole)Clmodified enzyme as electrochemical tag. (a) with capture probecomplementary to p53 gene (b) with capture probe non-complementary top53 gene. Conditions are the same as described in FIG. 3.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, Applicants have devised novelbiosensors and methods for detecting an analyte in a sample. The presentinvention is based on the synthesis of novel oxidation reductionmediators that can lose or gain electrons under various experimentalconditions. Without being held to a particular theory, it is believedthat such redox mediators act as electron relay groups that enablenon-diffusion-mediated electron transfer from other oxidation reductionmolecules such as, for example, oxidoreductases.

In various embodiments, the present invention provides transition metalcompounds that can function as redox mediators and have structures thatcorrespond to formula I described above wherein M is a metallic elementthat can form a coordinate bond to nitrogen. Suitable metallic elementsfor use as M can be, for example, osmium (Os), ruthenium (Ru), zinc(Zn), iron (Fe), rhodium (Rh), rhenium (Re), platinum (Pt), scandium(Sc), titanium (Ti), vanadium (V), cadmium (Cd), magnesium (Mg), copper(Cu), cobalt (Co), palladium (Pd), chromium (Cr), manganese (Mn), nickel(Ni), Molybdenum (Mo), tungsten (W), iridium (Ir) and mixtures thereof.In a preferred embodiment, the metallic element M is the transitionmetal osmium (Os).

R and R′ can be the same or different and are coordinated to M at theirnitrogen atoms. R, R′, or both can be, for example, 2,2′-bipyridyl;2,2′-bipyridyl substituted with one or more substituents selected fromthe group consisting of C1-C4 alkyl, phenyl and phenyl substituted withone or more C1-C4 alkyl groups; 1,10-phenanthrolinyl and1,10-phenanthrolinyl substituted with one or more substituents selectedfrom the group consisting of C1-C4 alkyl, phenyl and phenyl substitutedwith one or C1-C4 alkyl groups. Preferably, at least one of R and R′ is2,2′-bipyridyl.

L is a linking ligand. In various embodiments, L is an organic aminehaving from 3 to 20 non-hydrogen atoms, comprising an aliphatic aminogroup and further comprising a nitrogenous moiety that provides ametal-to-nitrogen coordinate bond to M. In a preferred embodiment, thenitrogenous moiety is a heterocyclic ring containing at least onenitrogen atom. Non-limiting examples include imidazole, benzimidazole,pyrroles, pyrazole, triazoles, benzotriazoles, pyridine, pyridazine,pyrazine, pyrimidine and triazines. One of the nitrogens of theheterocyclic ring forms a coordinate to the metal M. A preferrednitrogenous moiety is imidazole. When the nitrogenous moiety is aheterocyclic ring, the aliphatic amino group is preferably held on analkyl group attached to the ring. The alkyl group may be straight chainor branched and contains generally from 1 to about 20 carbons,preferably from 2 to 12 and more preferably from 3 to 6 carbon atoms. Apreferred linking ligand L is 3-aminopropylimidazole.

Z is a halogen atom. In a preferred embodiment Z is chlorine or bromine,more preferably chlorine. The superscript m can be +1, +2, +3, +4, +5,or +6, depending on the oxidation state of the metal M. In a preferredembodiment, for example when the metal M is osmium in the +4 oxidationstate, Z is chlorine and m is +3. X is an anion, or combination ofanions, that balances the formal charge m of the cation. For example, Xcan be, without limitation, chloride, bromide, iodide, fluoride,tetrafluoroborate, perchlorate, nitrate, sulfate, carbonate, or sulfite.

In other embodiments, at least one and preferably both, of the ligandinggroups R and R′ is a 2,2′-bipyridyl or 1,10-phenanthrolinyl, either ofwhich can be optionally substituted. When the bipyridyl orphenanthrolinyl is substituted, the substituents are preferably selectedfrom among C1 to C4 alkyl groups, phenyl groups and phenyl groupssubstituted further with C1-C4 alkyl, especially C1-C2 alkyl groups. Thesubstituted bipyridyl and phenathrolinyl liganding groups can bemonosubstituted, disubstituted, or higher substituted. In variousembodiments, disubstituted liganding groups can be used. Non-limitingexamples include 4,4′-disubstituted-2,2′-bipyridyl,5,5′-disubstituted-2,2′-bipyridyl, 1,10-phenanthrolinyl,4,7-disubstituted-1,10-phenanthrolinyl and5,6-disubstituted-1,10-phenanthrolinyl.

When only one of R and R′ is a bipyridyl or phenanthrolinyl or one ofthe optionally substituted groups discussed above, the other ispreferably selected from aliphatic ligands containing two nitrogen atomscapable of forming coordinate bonds with the metal M. Non-limitingexamples include 1,3-propanediamine, 1,4-butanediamine and derivativesof either, where the derivatives are based on 1,3-propanediamine or1,4-butanediamine skeletons optionally substituted with alkyl, aryl, orother groups that do not interfere with coordinate bonding of thenitrogens to the metal M or with the electrochemical activity of thecomplex.

In preferred embodiments, the transition metal compounds have one ormore of the following characteristics: a suitable redox potential tooperate as an electron shuttle in the biosensors of the invention, thepotential generally being intermediate between that of the prostheticgroup of the biomolecule and the biosensor electrode potential; theability to exchange electrons rapidly with electrodes, the ability torapidly transfer electrons to or rapidly accept electrons from an enzymeto accelerate the kinetics of electrooxidation or electroreduction of asubstrate in the presence of an enzyme or another electrochemicallyactive redox complex catalyst.

In a preferred embodiment, the transition metal compound comprises acation with a formal +3 charge having the structure:

The invention also provides for methods of preparing transition metalcompounds that can function as redox mediators having a structure thatcorresponds to formula I described above. In a preferred embodiment ofthe method, the transition metal compound is formed by ligand exchangeof a precursor compound of general formula II above with a linkingligand. The transition metal compounds described herein comprise fillerligands Z that help form a stable complex with the metal and aredisplaceable by the linking ligand under the conditions of the ligandexchange. Preferred linking ligands comprise a nitrogenous moiety thatprovides a metal-to-nitrogen coordinate bond to the metal. In apreferred embodiment, the metal is osmium, Z is chlorine and the linkingligand is 3-aminopropylimidazole.

In another embodiment, the transition metal compounds are used as redoxmediators as part of an electrochemical tag. The electrochemical tag isobtained when the transition metal compound is attached to a redoxbiomolecule via the linking ligand. In various embodiments, thetransition metal compound is covalently attached to amino acids of theredox biomolecule to form the electrochemical tag. Preferably, thetransition metal compound is attached to the amino acid residues of theredox biomolecule that are in relative proximity to the active-siteprosthetic groups of the redox biomolecule. In a preferred embodiment,the transition metal compound is attached to glucose oxidase ataspartate and glutamate residues. Computer graphic analysis shows thatat least two glutamate and eight aspartate residues are within at least16 Angstroms from the flavin adenine dinucleotide (FAD) N5 atom of theFAD/FADH prosthetic groups within the 160 kiloDalton glucose oxidasehomodimer.

In a preferred embodiment, the present invention provides for anelectrochemical tag comprising a redox mediator that is a transitionmetal compound of general formula I and a redox biomolecule, for examplean oxidoreductase. In a particularly preferred embodiment, the metal ofthe transition metal compound is osmium and the linking ligand is3-aminopropylimidazole. In a preferred embodiment, the electrochemicaltag comprises a redox mediator that is a transition metal compound offormula I covalently attached to glucose oxidase.

In further embodiments, the present invention provides methods ofcovalently attaching the transition metal compound to a redoxbiomolecule to form the electrochemical tag. In a preferred embodiment,the transition metal compound is attached to the redox biomolecule bycarbodiimide coupling using, for example,1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) andN-hydroxysulfosuccinimide (NHS), which are known to those skilled in theart. Without being bound by theory, carbodiimide catalyzes the formationof amide bonds between carboxylic acids or phosphates and amines byactivating carboxyl or phosphate to form an O-urea derivative. Thisderivative can react readily with nucleophiles. The reagent can be usedto make ether links from alcohol groups and ester links from acid andalcohols or phenols and peptide bonds from acid and amines. Carbodiimideis often used in the synthesis of peptides as the water-solublederivative EDC or as the organic soluble derivative,N,N′-dicyclohexyl-carbodiimide (DCC). NHS is often used to assist thecarbodiimide coupling in the presence of EDC. The reaction can includeformation of the intermediate active ester (the product of condensationof the carboxylic group and N-hydroxysuccinimide) that further reactswith the amine function to yield finally the amide bond.

The invention also provides, in various embodiments, methods for usingthe electrochemical tag to electrochemically determine an analyte in asample. Without limiting the mechanism, function or utility of presentinvention, it is believed that such methods take advantage of theefficient electron transfer from the electrochemical tag to theelectrodes of the biosensors of the invention. The methods of thepresent invention can be applied to the determination of a wide varietyof analytes in a wide range of samples.

In a non-limiting example, an electrochemically active redox complexcomprising the electrochemical tag is formed at the surface of a solidsupport, such as an electrode and electron transfer is detected. In oneembodiment, the complex is formed at the surface by virtue of attractionto or binding of analyte with a probe that either is immobilized on theelectrode surface or is able to bind to the electrode surface. Thebinding of analyte to probe depends on the analyte (the “target”) to bedetected. For example, where the analyte is nucleic acid, a suitableprobe is a nucleic acid comprising a single-stranded regioncomplementary to a specific sequence of the analyte. The analyte to bedetermined can comprise nucleic acid, which may be single stranded ordouble stranded, as specified, or contain portions of both doublestranded or single stranded sequence. Where the analyte comprises onlydouble stranded nucleic acid, it is understood that strand separation isrequired prior to hybridization of the analyte to the complementaryprobe. The analyte may be DNA (either genomic or cDNA), RNA, or ahybrid, where the nucleic acid contains any combination of deoxyribo-and ribo-nucleotides and any combination of bases, including uracil,adenine, thymine, cytosine, guanine, inosine, xathanine andhypoxathanine, etc. Where the analyte to be detected comprises anantigen, a suitable probe is an antibody that specifically recognizesthe antigen. Alternatively, where the target analyte is an antibody, theprobe can comprise an antigen recognized by the antibody. The binding ofanalyte to probe can be, in a non-limiting example, by hybridization,annealing, charge-charge interaction, hydrophobic interaction, orcovalent bonding. Generally, the probes can comprise, for example,oligonucleotides, including DNA, mRNA, rRNA, tRNA, peptide nucleic acids(PNAs), expressed sequence tags (ESTs), antigen, antibody, ligand orreceptor.

The interaction between the electrochemical tag and the analyte isachieved utilizing a variety of recognition pairs that are known tothose skilled in the art. In a non-limiting example, a recognition pairconsists of biotin and avidin. To use the recognition pair, therespective members of the pair are covalently attached to the analyteand the electrochemical tag. Specific interaction of the two members ofthe pair causes a complex to be formed that contains the two chemicalspecies and the covalently bound recognition pairs.

Thus, in one embodiment, the complex is formed at the electrode surfaceby interaction of immobilized probe on the electrode surface, analytelabeled with a first member of a recognition pair and electrochemicaltag labeled with a second member of the recognition pair. The labeledcomponents are combined in solution with an oxidation substrate of theredox biomolecule and the solution placed in contact with the electrode.The electrode is operated at a suitable potential to efficiently acceptthe electrons from the oxidation substrate by way of the metal compoundcovalently attached to the redox biomolecule as described above. Theprobe and analyte specifically bind or hybridize to each other, asdiscussed above. Since the probe is immobilized onto the surface of theelectrode, hybridization of the analyte onto the probe results inbringing the electrochemical tag into proximity to the electrode, whereelectron transfer can occur.

In preferred embodiments, the methods are used in genetic diagnosis. Forexample, oligonucleotide probes can be used for determining targetanalyte sequences such as the gene for p53, which is a gene associatedwith a variety of cancers. Other non-limiting examples include the genefor nonpolyposis colon cancer, the BRCA1 breast cancer gene, the Apo E4gene that indicates a greater risk of Alzheimer's disease allowing foreasy presymptomatic screening of patients, mutations in the cysticfibrosis gene, or any of the others well known in the art.

In one preferred embodiment, direct amperometric detection of target p53gene is performed using mRNA extracted from rat liver using anelectrochemical tag comprising an avidin conjugated-glucose oxidasecovalently attached to the transition metal compound. Utilizingtechniques known in the art, the mRNA is optionally further prepared byconverting the mRNA into cDNA, which is then labeled with biotin. Anelectrochemically active redox complex is formed at the electrodesurface where probe is bound to the electrode, analyte is bound to probevia nucleic acid hybridization and electrochemical tag is bound toanalyte via recognition pair binding involving avidin and biotin,respectively. As explained elsewhere, those skilled in the art willrecognize that one is not limited to the use of biotin/avidinrecognition pairs and that a variety of recognition pairs exist that canbe utilized in accordance with the present invention. Thus theelectrochemical tag is brought to the electrode surface following targetanalyte hybridization. In the presence of substrate (in this caseglucose), the electrochemical tag comprising the redox biomolecule (inthis case glucose oxidase) catalyzes the oxidation of substrate and theelectrons are shuttled to the electrode surface by the transition metalcompound attached to the redox biomolecule backbone. The determinationor detection of analyte is determined by detection of electron transferbetween the electrochemically active redox complex and the electrode. Acurrent is detected when the shuttled electrons are picked up by theelectrode.

In a further embodiment, the analyte to be detected is also theoxidation substrate for the redox biomolecule. In a non-limitingexample, the redox biomolecule component of the electrochemical tagcomprises glucose oxidase and the analyte is glucose. The presence ofthe oxidation substrate is detected as electron transfer activity at theelectrode (detection of current or voltage), where the electrochemicaltag is held or formed at the electrode surface utilizing techniquesknown in the art. In a non-limiting example, the surface of theelectrode is labeled with a first member of a recognition pair and theelectrochemical tag is labeled with a second member. The labeledcomponents can be combined in solution with the sample containing theanalyte to be detected (in this case, for example, glucose), which isalso the oxidation substrate of the redox biomolecule. Binding of themembers of the recognition pair to each other results in bringing theelectrochemical tag into proximity to the electrode, where electrontransfer can occur. The electrochemical tag comprising glucose oxidasecatalyzes the oxidation of any glucose present in the sample and theelectrons are shuttled to the electrode surface by the transition metalcompound attached to the glucose oxidase backbone. The determination ordetection of glucose is determined by detection of electron transferbetween the electrochemical tag and the electrode. A current is detectedwhen the shuttled electrons are picked up by the electrode.

In a preferred embodiment, probe is attached to gold electrodes.However, one skilled in the art knows that the probe can be immobilizedto the electrode by a number of techniques known in the art. Forexample, recognition pairs can be used to attach probe to electrode. Inthis regard, the probe can be modified to comprise a first member of arecognition pair where the electrode surface is coated with a secondmember, the recognition pair being distinct from the recognition pairused for binding of analyte to the electrochemical tag. In furtherexamples, the probes of the present invention can be covalently attachedto thin film oxidized surfaces. Employing literature procedures, avariety of techniques are available and known to those skilled in theart for immobilizing probes onto the surface of electrodes for use inaccordance with the present invention.

In various other embodiments, viral and bacterial detection can becarried out using the complexes of the invention. In this embodiment,probes are designed to detect target sequences from a variety ofbacteria and viruses. The methods disclosed herein allow for directscreening of clinical samples to detect, for example, HIV nucleic acidsequences. In addition, this allows direct monitoring of circulatingvirus within a patient as an improved method of assessing the efficacyof anti-viral therapies. Similarly, viruses associated with leukemia,HTLV-1 and HTLV-II, may be detected in this way. Bacterial infectionssuch as tuberculosis may also be detected.

In other embodiments, nucleic acids are used as probes for toxicbacteria in the screening of, for example water and food samples. Forexample, samples may be treated to lyse the bacteria to release itsnucleic acid and then probes designed to recognize bacterial strains,including, but not limited to, such pathogenic strains as, Salmonella,Campylobacter, Vibrio cholerae, enterotoxic strains of E. coli andLegionnaire's disease bacteria. Similarly, bioremediation strategies maybe evaluated using the compositions of the invention.

In other embodiments, the probes can be used for forensics where DNAfingerprinting is used to match crime-scene analytes such as DNA againstsamples taken from victims and suspects.

The source of analyte can include, for example, humans, animals, plants,or environment.

In other embodiments, the present invention is also directed to kitscomprising electrochemical tags. The kits can further comprise probes,such as those described herein, which can recognize and bind to theanalyte to be detected.

In various other embodiments, the present invention is further directedto a biosensor that utilizes the electrochemical tags described herein.The biosensor may comprise an apparatus or be used in a system thatincludes the necessary components for detecting and measuring a signalproduced by one or more electrochemical tags. An apparatus can compriseintegrated circuits including a biosensor array combined with a powersupply and a detector. Such integrated circuits are known to those ofskill in the art. Systems including the biosensor array may additionallyinclude means for measuring an electrochemical signal after a potentialis applied across a working electrode. Applying the electrical potentialand measuring the electrochemical signal can be accomplished with aprogrammed processor. The signal to be detected can be, for example,measured by pulse amperometry, by intermittent pulse amperometry, ordifferential pulse amperometry. Alternatively, the biosensor maycomprise a single working electrode and a single reference electrode.Whether in an array or a single working electrode, the biosensor mayoptionally include one or more counter electrodes.

The methods and apparatus described herein utilize laboratory techniqueswell known to skilled artisans and can be found in laboratory manualssuch as Sambrook, J., et al., Molecular Cloning: A Laboratory Manual,3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,2001; Spector, D. L. et al., Cells: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1998; and Harlow, E.,Using Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1999.

The headings (such as “Background of the Invention” and “Summary of theInvention”) used herein are intended only for general organization oftopics within the disclosure of the invention and are not intended tolimit the disclosure of the invention or any aspect thereof. Inparticular, subject matter disclosed in the “Background of theInvention” may include aspects of technology within the scope of theinvention and may not constitute a recitation of prior art. Subjectmatter disclosed in the “Summary of the Invention” is not an exhaustiveor complete disclosure of the entire scope of the invention or anyembodiments thereof.

The citation of references herein does not constitute an admission thatthose references are prior art or have any relevance to thepatentability of the invention disclosed herein. All references cited inthe specification are hereby incorporated by reference in theirentirety.

The description and specific examples, while indicating embodiments ofthe invention, are intended for purposes of illustration only and arenot intended to limit the scope of the invention. Moreover, recitationof multiple embodiments having stated features is not intended toexclude other embodiments having additional features, or otherembodiments incorporating different combinations of the stated features.Specific Examples are provided for illustrative purposes of how to make,use and practice the compositions and methods of this invention and,unless explicitly stated otherwise, are not intended to be arepresentation that given embodiments of this invention have, or havenot, been made or tested.

As used herein, the words “preferred” and “preferably” refer toembodiments of the invention that afford certain benefits, under certaincircumstances. However, other embodiments may also be preferred, underthe same or other circumstances. Furthermore, the recitation of one ormore preferred embodiments does not imply that other embodiments are notuseful and is not intended to exclude other embodiments from the scopeof the invention.

As used herein, the word “include,” and its variants, is intended to benon-limiting, such that recitation of items in a list is not to theexclusion of other like items that may also be useful in the materials,compositions, devices and methods of this invention.

In the following description the terms “determination” or “detection”will be used to denote both qualitative and quantitative determinationor detection of analyte. Where, for example, the method and systemdefined below are used for determining or detecting an analyte in aliquid medium, this is meant to denote determining the presence of ananalyte in the medium and optionally its concentration.

The term “analyte” as used herein and will be used further below, ismeant to denote an agent determined in a liquid sample. Analyte may ormay not be the substrate for a redox biomolecule.

The term “redox biomolecule” as used herein and will be used furtherbelow, is meant to denote biomolecules, native or otherwise modified orengineered, that are inherently capable of electron transfer, such asfor example, enzymes that catalyze the oxidation or reduction of asubstrate or a group of substrates. Some redox biomolecules useprosthetic groups, such as flavins or nicotinamide adenine dinucleotide(NAD). A prosthetic group is the non-protein component of the redoxbiomolecule that is bound to the redox biomolecule enabling it tocatalyze oxidation or reduction. The simplest redox biomolecules, inwhich no prosthetic group is present, are those that use reversibleformation of a disulfide bond between two cysteine residues, as inthioredoxin. Many use the ability of iron or copper ions to exist in twodifferent redox states.

The term “substrate” or “oxidation substrate” as used herein and will beused further below, is meant to denote a molecule that binds to a redoxbiomolecule active site and undergoes a reaction, such as oxidation orreduction.

The term “redox mediator” as used herein and will be used further below,is meant to denote molecules that are used to carry electrons betweenthe redox biomolecule and the electrode.

The term “electrochemical tag” as used herein and will be used furtherbelow, is meant to denote a redox biomolecule covalently attached to aredox mediator.

The term “electrochemically active redox complex” as used herein andwill be used further below, is meant to denote a complex formed at thesurface of an electrode comprising the electrochemical tag.

The term “biosensor” as used herein and will be used further below, ismeant to denote an apparatus or system that comprises the necessarycomponents for detecting or measuring a signal produced by movement ofelectrons produced in an oxidation and reduction reaction (for example,amperometric detection). The term “biosensor” includes devices fordetermining the concentration of substances and other parameters ofbiological interest even where a biological system is not directlyutilized.

EXAMPLES

The following examples are intended to be illustrative and are notintended to limit the scope of the invention.

Example 1

This example illustrates the synthesis of the redox mediator,Os(bpy)₂(3-aminopropylimidazole)Cl.

Os(bpy)₂Cl₂ is synthesized from K₂OsCl₆ (99%, Stem Chemicals) followingthe proposed procedure as described, for example, in Lay, P. A. et al.,1986, Inorg. Synth., 24: 291-296. Os(bpy)₂(3-aminopropylimidazole)Cl issynthesized as follows: To a solution of Os(bpy)₂Cl₂ (0.10 mmol) in 6.0ml fresh-distilled ethylene glycol is added 3-aminopropylimidazole (0.12mmol) in one portions the result mixture is refluxed for 90-120 min.3-aminopropylimidazole was purchased from Sigma-Aldrich (St. Louis,Mo.). The completion of the ligand-exchange reaction is monitored bycyclic voltammetry. The purple reaction mixture is then poured slowlyinto 200 ml of rapid stirred ether. The precipitate is collected bysuction filtration through a fine fritted funnel. The crude product iswashed with ether, dissolved in 3.0-5.0 ml of ethanol and precipitatedagain from ether. The precipitate is further purified by crystallizationfrom ethanol giving the pure product in 75% yield.

The product shows a single pair of reversible redox waves at a goldelectrode with an E_(1/2) of 0.19 V in phosphate buffered saline (PBS).To ensure a complete ligand-exchange, slight excess of3-aminopropylimidazole (10-20%) is required.

Cyclic voltammetry is used to monitor the course of the reaction. Thepurified product is characterized by mass spectrometry andelectrochemistry, which are consistent with the structure of:

Example 2

This example illustrates the modification of the redox biomolecule,glucose oxidase (GOx), by coupling to a redox mediator.

Glucose oxidase-avidin D conjugate (GOx-A) (131 units/mg of solid) waspurchased from Vector Laboratories (San Diego, Calif.). Glucose oxidase(GOx, EC 1.1.3.4, type X-S, from Aspergillus niger, 213 units/mg ofsolid) was purchased from Sigma-Aldrich (St. Louis, Mo.).1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC),N-hydroxysulfosuccinimide (Sulfo-NHS) and dialysis kits (MWCO 10,000)were obtained from Pierce.

GOx is covalently modified with Os(bpy)₂(3-aminopropylimidazole)Cl byamide bond formation between GOx carboxylates and the aliphatic primaryamino groups present on the redox mediators (e.g. transition metalcompound) with EDC/NHS as coupling agent. Excess redox mediator is usedto avoid protein self-crosslinking.

To 4.5 ml of 1.0 mM Os(bpy)₂(3-aminopropylimidazole)Cl and 1.0 Mavidin-conjugated glucose oxidase (GOx-A) in DI water, is added1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) andN-hydroxysuccinimide (NHS), yielding final concentrations of 4.0 mM EDCand 1.6 mM sulfo-NHS. The mixture is stirred for 2 h at roomtemperature. The solution is then purified by dialysis against PBSbuffer for 24 h. In a control experiment, same amount of GOx-A is mixedwith Os complex without adding coupling agent.

Example 3

This example illustrates that the electrochemical tag of GOx modified bycovalent attachment of the transition metal compoundOs(bpy)₂(3-aminopropylimidazole)Cl is electrochemically active.

Electrochemical experiments are carried out using a CH Instruments model660A electrochemical workstation coupled with a low current module (CHInstruments, Austin, Tex.). The three-electrode system consists of a2-mm-diameter gold working electrode, a miniature Ag/AgCl referenceelectrode (Cypress Systems, Lawrence, Kans.) and a platinum wire counterelectrode. Potentials are referred to the Ag/AgCl reference electrode.Cyclic voltammetry (CV) of the electrochemical tag shows a pair ofwell-defined voltammetric current peaks centered at 0.19 V, showingelectrochemical activity. To prove that the enzyme was indeed covalentlymodified, as opposed to simple electrostatic association withOs(bpy)₂(3-aminopropylimidazole)Cl, a control experiment is performedaccording to example 2 but in the absence of the EDC and NHS couplingreagents. The control experiment yields biologically active enzyme butwith no detectable mediator following dialysis, indicating that themediator is not covalently attached to GOx (curve b in FIG. 1).

Example 4

This example illustrates characterization of GOx modified withOs(bpy)₂(3-aminopropylimidazole)Cl using UV-vis absorbance spectra.

Uv-vis absorbance spectra of the starting materials and the modified GOxare depicted in FIG. 2. UV-vis spectrumOs(bpy)₂(3-aminopropylimidazole)Cl is similar to that of Os(bpy)₂compound. It exhibits intense band in the UV region due to intraligand(IL)σ→σ*(bpy) transitions and by a broad band in the visible region(400-600 nm) due to spin allowed Os(dπ)→bpy(π*) metal-to-ligandcharge-transfer (MLCT) transition. Moreover, the spectrum of themodified GOx is a composite of the absorbance spectra from both the GOxand Os(bpy)₂Cl(3-aminopropylimidazole) complex. A simple overlay ofOs(bpy)₂Cl(3-aminopropylimidazole) and GOx generates a spectrum which issimilar to that of the activated GOx, confirming the formation of theactivated GOx.

Example 5

This example illustrates the biological activity of GOx modified withOs(bpy)₂(3-aminopropylimidazole)Cl using UV-vis absorbance spectra.

To determine whether enzyme is still biologically active after thecovalent modification, one drop of the modified enzyme solution isapplied to a screen-printed carbon electrode. After 5-10 min adsorption,the electrode is thoroughly rinsed with PBS buffer. Catalytic current ismonitored by measuring the current at 0.3 V in the presence of 40 mMglucose solution in PBS. Amperometric results of immobilizedelectroactive enzyme reveal that the GOx retains its catalytic activitytoward the oxidation of glucose (FIG. 3). The catalytic current iscomparable to or better than that of native GOx at same concentration inthe presence of the best soluble mediator. Amperometric results furtherprove that the Os(bpy)₂(3-aminopropylimidazole)Cl covalently attached tothe GOx could promote direct reduction of the FAD centers of the enzymewithout the need of any additional mediator in solution. Similarly,GOx-Avidin conjugates modified with Os(bpy)₂(3-aminopropylimidazole)Clshow similar electrochemical properties as those of the activated GOx.

Example 6

This example illustrates the application of GOx-A modified withOs(bpy)₂(3-aminopropylimidazole)Cl as an electrochemical tag in nucleicacid detection.

The electrochemically activated GOx-A is used as an electrochemical tagin direct amperometric detection of nucleic acid. FIG. 4 shows a typicalamperometric curve for the detection of p53 gene in mRNA (20 pg/μl)extracted from rat liver (mRNA is converted to cDNA, which is thenlabeled with biotin). Nucleic acid preparation, capture probeimmobilization and measurements were essentially as described, forexample, in Xie, H. et al., 2004, Nucleic Acids Research, 32: e15. Whencomplementary probe is immobilized on the electrode surface, the enzymeis brought to the surface following target gene hybridization becausethe avidin moiety of GOx-A binds to the biotin moiety of the target. Inthe presence of glucose, the enzyme catalyzes the oxidation of glucose.The electrons are then being shuttled to the electrode surface byOs(bpy)₂(3-aminopropylimidazole)Cl attached to the GOx-A backbone andpicked up by the electrode. In a control experiment withnon-complementary capture probe on the electrode surface, the target p53gene fails to hybridize to the capture probe and hence enzyme is unableto bind onto the surface. The small current being detected is mainly dueto non-specific binding of enzyme. Table 1 gives the results of threeduplicated experiments.

TABLE 1 Amperometric response of p53 assay (3 replicates) 1 2 3 Sample2.53 nA 2.50 nA 2.34 nA Control 0.60 nA 0.46 nA 0.43 nA

As various changes could be made in the above methods and compositionswithout departing from the scope of the present teachings, it isintended that all matter contained in the above description beinterpreted as illustrative and not in a limiting sense. Illustrationsand examples are not intended to be a representation that givenembodiments of this present teachings have, or have not, been performed.

1-20. (canceled)
 21. A transition metal compound of the formula:

wherein M is a metallic element that can form a coordinate bond tonitrogen; R and R′ are independently selected from the group consistingof 1,10-phenanthrolinyl and 1,10-phenanthrolinyl substituted with one ormore substituents selected from the group consisting of C₁-C₄ alkyl,phenyl and phenyl substituted with one or more C₁-C₄ alkyl groups; n isan integer of 1 to 20, inclusive; Z is a halogen atom; m is +1, +2, +3,+4, +5, or +6; and X is an anion, or combination of anions, thatbalances m.
 22. The transition metal compound of claim 21, wherein M isselected from the group consisting of osmium, ruthenium, zinc, iron,rhodium, rhenium, platinum, scandium, titanium, vanadium, cadmium,magnesium, copper, cobalt, palladium, chromium, manganese, nickel,molybdenum, tungsten and iridium, or mixtures thereof.
 23. Thetransition metal compound of claim 22, wherein M is osmium.
 24. Thetransition metal compound of claim 21, wherein Z is chloro or bromo. 25.The transition metal compound of claim 21, wherein R and R′ are1,10-phenanthrolinyl; M is osmium; Z is chloro; and n is
 3. 26. A methodfor preparing a transition metal compound, comprising contacting aprecursor compound with a linking ligand to form a transition metalcompound of claim 21, wherein the precursor compound has the formula:

wherein M is a metallic element that can form a coordinate bond tonitrogen; R and R′ are independently selected from the group consistingof 1,10-phenanthrolinyl and 1,10-phenanthrolinyl substituted with one ormore substituents selected from the group consisting of C₁-C₄ alkyl,phenyl and phenyl substituted with one or more C₁-C₄ alkyl groups; Z isa halogen atom; m is +1, +2, +3, +4, +5, or +6; and X is an anion, orcombination of anions, that balances m.
 27. The method of claim 26,wherein M is osmium.
 28. The method of claim 26, wherein Z is chloro orbromo.
 29. The method of claim 26, wherein R and R′ are1,10-phenanthrolinyl; M is osmium; m is +3; Z is chloro; and the linkingligand is 3-aminopropylimidazole.
 30. An electrochemical tag comprisinga redox biomolecule bonded to a transition metal compound of theformula:

wherein M is a metallic element that can form a coordinate bond tonitrogen; R and R′ are independently selected from the group consistingof 1,10-phenanthrolinyl and 1,10-phenanthrolinyl substituted with one ormore substituents selected from the group consisting of C₁-C₄ alkyl,phenyl and phenyl substituted with one or more C₁-C₄ alkyl groups; n isan integer of 1 to 20, inclusive; Z is a halogen atom; m is +1, +2, +3,+4, +5, or +6; and X is an anion, or combination of anions, thatbalances m.
 31. The electrochemical tag of claim 30, wherein the redoxbiomolecule comprises an oxidoreductase.
 32. The electrochemical tag ofclaim 31, wherein the oxidoreductase comprises glucose oxidase.
 33. Theelectrochemical tag of claim 30, wherein M is osmium.
 34. Theelectrochemical tag of claim 30, wherein Z is chloro or bromo.
 35. Theelectrochemical tag of claim 30, wherein R and R′ are1,10-phenanthrolinyl; M is osmium; Z is chloro; n is 3; and the redoxbiomolecule comprises glucose oxidase.
 36. A method for preparing anelectrochemical tag of claim 30, comprising bonding a redox biomoleculeto a transition metal compound of the formula:

wherein M is a metallic element that can form a coordinate bond tonitrogen; R and R′ are independently selected from the group consistingof 1,10-phenanthrolinyl and 1,10-phenanthrolinyl substituted with one ormore substituents selected from the group consisting of C₁-C₄ alkyl,phenyl and phenyl substituted with one or more C₁-C₄ alkyl groups; n isan integer of 1 to 20, inclusive; Z is a halogen atom; m is +1, +2, +3,+4, +5, or +6; and X is an anion, or combination of anions, thatbalances m.
 37. The method of claim 36, wherein the redox biomoleculecomprises an oxidoreductase.
 38. The method of claim 37, wherein theoxidoreductase comprises glucose oxidase.
 39. The method of claim 36,wherein the redox biomolecule is bound to the transition metal compoundby an amide bond.
 40. The method of claim 36, wherein M is osmium. 41.The method of claim 36, wherein Z is chloro or bromo.
 42. The method ofclaim 36, wherein R and R′ are 1,10-phenanthrolinyl; M is osmium; Z ischloro; n is 3; and the redox biomolecule comprises glucose oxidase. 43.The method of claim 42, wherein the redox biomolecule is bound to thetransition metal compound by an amide bond.
 44. An electrochemicalmethod for detecting an analyte in a sample, comprising: forming at anelectrode surface an electrochemically active redox complex comprisingthe analyte and an electrochemical tag; and detecting electron transferat the electrode, wherein the electrochemical tag comprises a redoxbiomolecule bonded to a transition metal compound of the formula:

wherein M is a metallic element that can form a coordinate bond tonitrogen; R and R′ are independently selected from the group consistingof 1,10-phenanthrolinyl and 1,10-phenanthrolinyl substituted with one ormore substituents selected from the group consisting of C₁-C₄ alkyl,phenyl and phenyl substituted with one or more C₁-C₄ alkyl groups; n isan integer of 1 to 20, inclusive; Z is a halogen atom; m is +1, +2, +3,+4, +5, or +6; and X is an anion, or combination of anions, thatbalances m.
 45. The method of claim 44, wherein the analyte is anoxidation substrate for the redox biomolecule.
 46. The method of claim44, wherein the redox biomolecule comprises an oxidoreductase.
 47. Themethod of claim 46, wherein the oxidoreductase comprises glucoseoxidase.
 48. The method of claim 44, wherein M is osmium.
 49. The methodof claim 44, wherein Z is chloro or bromo.
 50. The method of claim 44,wherein R and R′ are 1,10-phenanthrolinyl; M is osmium; Z is chloro; andn is
 3. 51. The method of claim 44, wherein the method furthercomprises: immobilizing a probe onto the electrode surface; labeling theanalyte with a first member of a recognition pair; labeling theelectrochemical tag with a second member of the recognition pair; andcombining the labeled components with an oxidation substrate of theredox biomolecule in solution in contact with the electrode, wherein theprobe and analyte specifically bind or hybridize to each other.
 52. Themethod of claim 51 wherein the recognition pair comprises biotin andavidin.
 53. The method of claim 51, wherein the analyte comprises anucleic acid.
 54. The method of claim 44, wherein the method furthercomprises: labeling the electrode surface with a first member of arecognition pair; labeling the electrochemical tag with a second memberof the recognition pair; and combining the labeled components with anoxidation substrate of the redox biomolecule in solution in contact withthe electrode, wherein the electrochemical tag binds to the electrodesurface.
 55. The method of claim 54, wherein the recognition paircomprises biotin and avidin.
 56. The method of claim 55, wherein theanalyte comprises the substrate of the redox biomolecule.